Chitin, a β-1,4-linked un-branched polymer of N-acetylglucosamine (GlcNAc), constitutes the second most abundant polymer on earth following cellulose. It is a major component of insect exoskeletons (Merzendorfer, H., et al., J. Exptl. Biol. 206:4393-4412 (2003), the shells of invertebrate crustaceans and of fungal cell walls (Riccardo, A., et al. “Native, industrial and fossil chitins,” in Chitin and Chitinases, ed. P. Jolles and R. A. A. Muzzarelli, pub. Birkhauser Verlag: Basel, Switzerland (1999)). Chitinases hydrolyze the β-1,4-glycosidic bond of chitin and have been found in prokaryotic, eukaryotic and viral organisms. In the yeast Saccharomyces cerevisiae, chitinase plays a morphological role in efficient cell separation (Kuranda, M., et al. J. Biol. Chem. 266:19758-19767 (1991)). Additionally, plants express chitinases in defense against chitin-containing pathogens. In fact, the heterologous expression of chitinase genes in transgenic plants has been shown to increase resistance to certain plant pathogens (Carstens, M., et al. Trans. Res. 12:497-508 (2003); Itoh, Y., et al. Biosci. Biotechnol. Biochem. 67:847-855 (2003); Kim, J. et al. Trans. Res. 12:475-484 (2003)). Chitinases belong to either family 18 or family 19 of glycosylhydrolases based on their amino acid sequence similarities (Henrissat, B., et al. Biochem. J. 293:781-788 (1993)). Familial differences in chitinase catalytic domain sequences reflect their different mechanisms of chitin hydrolysis that result in either retention (family 18) or inversion (family 19) of the anomeric configuration of the product (Robertus, J. D., et al. “The structure and action of chitinases,” in Chitin and Chitinases, ed. P. Jolles and R. A. A. Muzzarelli, pub. Birkhauser Verlag: Basel, Switzerland (1999)).
Most chitinases have a modular domain organization with distinct catalytic and non-catalytic domains that function independently of each other. An O-glycosylated Ser/Thr-rich region often separates the two domains and may serve to prevent proteolysis or aid in secretion of the chitinase (Arakane, Y., Q. et al. Insect Biochem. Mol. Biol. 33:631-48 (2003)). Non-catalytic chitin binding domains (CBDs also referred to as ChBDs) belong to one of three structural classes (type 1, 2 or 3) based on protein sequence similarities (Henrissat, B. “Classification of chitinases modules,” in Chitin and Chitinases, ed. P. Jolles and R. A. A. Muzzarelli, pub. Birkhauser Verlag Basel, Switzerland (1999)). Depending on the individual chitinase, the presence of a CBD can either enhance (Kuranda, M., et al. J. Biol. Chem. 266:19758-19767 (1991)) or inhibit (Hashimoto, M., et al. J. Bacteriol. 182:3045-3054 (2000)) chitin hydrolysis by the catalytic domain.
The small size (˜5-7 kDa), substrate binding specificity and high avidity of CBDs for chitin has led to their utilization as affinity tags for immobilization of proteins to chitin surfaces (Bernard, M. P., et al. Anal. Biochem. 327:278-283 (2004); Ferrandon, S., et al. Biochim. Biophys. Acta. 1621: 31-40 (2003)). For example, the B. circulans chitinase A1 type 3 CBD has been used to immobilize fusion proteins expressed in bacteria on chitin beads to provide a platform for intein-mediated protein splicing (Ferrandon, S., et al. Biochim. Biophys. Acta. 1621: 31-40 (2003)) and to chitin-coated microtiter dishes (Bernard, M. P., et al. Anal. Biochem. 327:278-283 (2004)). Because eukaryotic protein expression systems are capable of biological processes not possible with bacterial systems (e.g. protein glycosylation, chaperone-mediated protein folding etc.) it is also desirable to secrete CBD-tagged proteins from eukaryotic cells. However, many eukaryotic cells, especially fungi, secrete endogenous chitinases that complicate the immobilization of CBD-tagged proteins to chitin by competing with the CBD-tagged protein for chitin-binding sites, by co-purifying with the CBD-tagged protein during chitin immobilization applications, and by degrading the target chitin-coated surface.
Proteins secreted from host cells into the surrounding media are substantially diluted resulting in a costly and cumbersome purification from large volumes. It is desirable to reduce the cost and increase the ease of separating proteins from the media in which they are secreted.
A variety of approaches exist to purify proteins from large volumes of media. These approaches vary in cost, efficiency and length of time required to achieve purification. For example, proteins in secreted culture can be harvested by precipitation. This approach requires addition of large quantities of a precipitating agent such as ammonium sulfate, acetone, or trichloroacetic acid, followed by centrifugation or filtration. Many of these agents are toxic or volatile, and all add significant expense to protein harvesting. Additionally, precipitation can result in significant loss of protein function.
Another approach is chromatography using various resins such as anion/cation exchange resins, hydrophobic interaction resins, or size exclusion gels. Harvesting proteins by chromatography requires that all of the spent culture medium be passed through the resin at a slow flow rate (typically, 1-10 ml min−1). This can be very time-consuming in instances where large volumes of medium must be processed. For example, 100 liters of spent culture medium passed through a resin at a 5 ml min−1 flow rate would take 333 hours to process. Additionally, these types of chromatography resins do not selectively purify only the target protein and must often be used in conjunction with other methods in a multi-step purification process.
Affinity chromatography resins that specifically bind peptide sequences incorporated into the protein's structure are often used because of their ability to selectively purify a target protein. In a typical strategy, a peptide sequence (e.g. a peptide antibody epitope or hexahistidine sequence) is engineered into the desired protein's sequence. A protein expressed with one of these tags will specifically interact with a corresponding resin (e.g. a resin having an immobilized antibody or a nickel resin for hexahistidine binding). While these methods often produce highly purified proteins from small volumes, they are limited in their practicality for processing large volumes by their cost and performance. For example, antibody affinity resins are very expensive and nickel resins can result in co-purification of undesired proteins that happen to contain stretches of histidine residues.
Magnetic techniques using magnetic carriers including beads have been used to purify proteins from cultures (Safarik et al. Biomagnetic Research and Technology 2:7 (2004)). A problem with this approach has been the need to customize each magnetic bead reagent to bind individual secreted proteins. This may involve complex chemistry to attach the affinity ligands to the beads. This also represents hurdles in efficiency and cost.
In some cases, natural affinities between the secreted protein and a substrate have been exploited. For example, lysozyme has a binding affinity for chitin so that when the hen egg white enzyme is exposed to chitin, it can be purified (Safarik et al. Journal of Biochemical and Biophysical Methods 27:327-330 (1993)).